Defensins are tridisulfide peptides implicated in innate immunity against potentially pathogenic microorganisms. Myeloid defensins are packaged in the granules of neutrophils and monocytes, and epithelial defensins are expressed in a wide variety of mucosal tissues. The most recently discovered defensins, termed theta-defensins, are 18-amino acid macrocyclic peptides that are stabilized by three parallel disulfide bonds. Isolated from rhesus monkey leukocytes, theta-defensins are remarkably potent antibiotics that kill bacteria and fungi, and they inactivate HIV-1. Antimicrobial activity is abrogated by opening of the backbone ring. The presence of macrocyclic peptides in animals was not previously known. Moreover, the biosynthesis of theta-defensins is novel, as the cyclic peptide is synthesized from two 9-amino acid segments that are spliced together in a head-to-tail configuration. While the cellular machinery that mediates this post-translational pathway is unknown, we hypothesize that enzymes expressed in theta-defensin-producing cells are responsible for the nonapeptide excision and ligation steps necessary for biosynthesis of the mature cyclic molecule. We propose to characterize the molecular components of the theta-defensin processing pathway by pursuing three specific Aims: 1. In Specific Aim 1, we will analyze the pro-theta-defensin intermediates produced in myeloid cells, and will determine the subcellular compartments of the molecular intermediates identified. 2. Specific Aim 2 is to identify pro-theta-defensin converting activities in extracts of theta-defensin-expressing cells. For these studies we will use synthetic and recombinant forms of putative substrates involved in the excision/ligation pathway, and use immunoprecipitation, and chromatographic, electrophoretic, and mass spectroscopic methods for detecting and characterizing the relevant enzymatic activities. 3. Specific Aim 3 is to characterize proteins that interact with pro-theta-defensins and subsequent intermediates, as these are likely to be convertases or chaperones necessary for the excision/ligation steps involved in theta-defensin biosynthesis. Results obtained from these studies are likely to disclose novel mechanisms that have evolved for splicing and cyclizing proteins in mammalian cells.